Nuclease free water

Two micrograms of small RNA from each sample was incubated with deacylation buffer (pH = 9.0) at 37 °C for 45 min and followed by ethanol precipitation. The small RNA was recovered in 20 µL nuclease-free water (Ambion). Then the recovered small RNA was treated with TURBO DNA-free kit (Ambion) for DNA contamination removing. The small RNA was purified from the reaction by ethanol precipitation. Then the recovered small RNA was incubated with 1 U Cap-Clip Acid Pyrophosphatase (Cellscript) in 1× Cap-Clip Acid Pyrophosphatase reaction buffer (Cellscript) at 37 °C for 30 min. Then, the reaction was added with 20 U T4 PNK (NEB) in 1× T4 PNK reaction buffer (NEB) and 1 mM ATP (NEB) and incubated at 37 °C for 30 min. The small RNA was purified from the reaction by phenol-chloroform extraction and ethanol precipitation. Then the purified small RNA was treated with 2× molar ratio of AlkB and 4× molar ratio of AlkB (D135S) at 25 °C for 1 h with 300 mM KCl, 2 mM MgCl₂, 10 µM of (NH4)2Fe(SO4)2·6H₂O, 300 µM 2-ketoglutarate (2-KG), 2 mM L-ascorbic acid, 50 µg/ml BSA, 50 mM MES buffer (pH 5.0). The reaction was quenched by addition of 5 mM EDTA. After phenol-chloroform extraction and ethanol precipitation, the small RNA was recovered in 3 µL nuclease-free water (Ambion).

Positive samples for the irbp gene were submitted to a previously described [53 (link)] conventional (c) PCR protocol aiming to amplify 800 bp of the hemotrophic Mycoplasma spp. 16S rRNA gene. All PCRs were performed with nuclease-free water (Thermo Scientific©, Waltham, MA, USA) as a negative control. Mycoplasma wenyonii and ‘Candidatus Mycoplasma haemobos’ DNA samples from naturally infected cattle [54 (link)] were used as positive controls. The reaction mixture for the PCR Protocol was composed of 1.25 U of Taq DNA Polymerase (Thermo Scientific©, Waltham, MA, USA), 0.2 mM of each deoxynucleotide, 1.5 mM of MgCl₂, 0.5 µM of each primer, and 5 µL of template DNA brought to a total volume of 25 µL with nuclease-free water (Thermo Scientific©, Waltham, MA, USA). The thermic protocol is described in Table 1.
Conventional PCR results were visualized in 1% agarose gel stained by Ethidium bromide ultrapure solution (Life Technologies©, Carlsbad, CA, USA). 16S rDNA amplicons that presented a good band quality in the agarose gel were purified using a Silica Bead DNA Gel Extraction Kit (Fermentas, São Paulo-SP, Brazil), following the manufacturer’s instructions, and sent to the Center of Biological Resources and Genomic Biology (CREBIO, Jaboticabal, SP, Brazil) for sequencing by Sanger’s method with ABI PRISM 3700 DNA Analyzer (Applied Biosystems©, Foster city, CA, USA).

Formalin-fixed paraffin embedded tissues were sectioned into 20-micron sections and placed into sterilized, nuclease free tubes. Sections were treated and RNA isolated according to manufacturer’s instruction (RecoverAll, Thermo Scientific-Ambion). Recovered RNA was analyzed for integrity and quantified using a Bioanalyzer and RNA 6000 Nano Kit (Agilent). Amounts of RNA were diluted into 500 ng/µL using nuclease-free water (Thermo Scientific). Gene analysis was performed utilizing RT2 profiling array for bovine inflammatory genes per manufacturer’s instruction (Inflammatory Cytokines and Receptors Array Plate, Qiagen). Briefly, 500 ng of RNA (1 µL), 1 µL of nuclease free water (Thermo Scientific) and 18 µL of 1-step RT-qPCR reaction buffer (OneStep Kit, Qiagen) was added to each well. A BioRad CFX96 Real Time PCR Systems thermocycler (Bio-Rad, Hercules, CA) was used. Amplification conditions for were: 30 min 50 °C, 10 min at 95 °C, 40 cycles of 15 s 95 °C and 1 min 60 °C (measure florescence step) and a dissociation step of 15 s 95 °C, 1 min 60 °C, 15 s 95 °C, 15 s 60 °C. Ct values were calculated and normalized to the endogenous control and expressed relative to No-Adj/No-Vac tissue sections using the 2^−ΔΔCT method.

We ordered all DNA oligos from Integrated DNA Technologies (Coralville, IA, USA), and dissolved each into nuclease-free water (Thermo Fisher) prior to storage at −20°C. On the day of an experiment, we prepared stocks of each DNA complex in DEPC-treated phosphate-buffered saline (PBS) by slowly annealing the strands from 97°C to 23°C at a rate of −2°C/min. We then stored these DNA complexes on ice and protected from light until the time of experiment. We prepared LAMP primer mix stocks in nuclease-free water (Thermo Fisher) and stored at −20°C.